The Role of RNA Structure in the Hepatitis B Virus Lifecycle

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SUMMARY Globally, a chronic hepatitis B virus (HBV) infection is the most common cause of hepatocellular carci- noma (HCC). An estimated 240 million people are chronically HBV-infected worldwide, and approved treatments are rarely curative. HBV has a DNA genome enclosed in a virally encoded protein capsid surrounded by a protein/lipid envelope. After infection of a hepatocyte, the viral genome is delivered to the nucleus and converted to an episome referred to as covalently closed circular (ccc)DNA. cccDNA is not targeted by current anti-HBV therapies, and its stability is linked to viral rebound when these therapies are stopped. Attempts to target cccDNA have not succeeded, and there is a need for new anti-HBV therapies. cccDNA is not directly replicated but is the template for HBV RNA transcripts. One of the transcripts, the pregenomic (pg)RNA, is encapsidated and reverse- transcribed by the virally encoded reverse-transcriptase/polymerase (RT/Pol) to generate the HBV DNA genome. Recent advances in RNA structure probing techniques have led to the discovery of RNA structural elements in several viruses. Many of these elements have critical roles in the viral lifecycle, and targeting RNA structures in viral genomes could open new avenues of drug development. Despite the therapeutic potential, the role of RNA structure in the HBV lifecycle is largely unexplored. The multi-functional HBV pgRNA is the template for reverse transcription and is also translated to produce the core (capsid) and RT/Pol proteins. The RT/Pol open-reading frame (ORF) is 3’ of the core ORF on pgRNA and overlaps with the 3’ end of the core ORF; synthesis of RT/Pol requires internal initiation of translation by a mechanism that is not understood. RNA structures referred to as epsilon (ε) stem-loops are located near the 5’- and 3’-ends of pgRNA, yet only the 5’ ε is required for pgRNA encapsidation. Due to limitations of available techniques, prior studies focused on local regions in HBV RNA transcripts, and many questions remain. For example, whether encapsidated and translated pgRNAs are structurally different, how internal initiation of RT/Pol synthesis is regulated, and why only the 5’ ε affects pgRNA encapsidation are incompletely understood or entirely unknown. New techniques to examine RNA structure in living cells, such as SHAPE-MaP (selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling) and DMS-MaP (dimethyl sulfate probing and mutational profiling), couple RNA chemical probing techniques with next-generation sequencing to identify RNA structures over the entire length of an RNA transcript. We will use these techniques to define RNA structural elements in HBV pgRNA and the role of these elements in the HBV lifecycle. We hypothesize that pgRNA harbors structural elements that have critical roles in the HBV lifecycle, and our studies could identify novel and highly specific targets for blocking HBV replication and the development of HCC.